JP2007240528A - Acquisition device for information as to concentration of thioredoxins in sample, stress degree information acquisition device, and stress degree determination method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an information acquisition device for acquiring information as to at least one or more out of concentrations of oxidized forms and concentrations of reduced forms of thioredoxins, useful for determining a stress degree, and concentration ratios thereof, and a stress degree information acquisition device and a stress degree determination method using the same. <P>SOLUTION: At least the one or more out of the concentrations of the oxidized forms and the concentrations of the reduced forms of thioredoxins, and the concentration ratios thereof, is measured using a reaction by an enzyme or the like catalyzing a redox reaction for the thioredoxins, and the stress degree of a measuring objective person is determined using a data thereof. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an information acquisition device for acquiring information related to the concentration of thioredoxins, a stress sensor using the information, a stress level information acquisition device, and a stress level determination method. More specifically, the present invention relates to a measurement method for distinguishing oxidized and reduced forms of thioredoxin and measuring these concentrations, an apparatus for obtaining information on the concentration of thioredoxins using this measurement method, and use thereof.

  There are many types of stress that individuals and cells receive. Moreover, the mechanism of the response to them has elements common to each other, and many signal pathways are activated at the same time and are regulated in close relation. Among these stresses, oxidative stress occupies an important position among stresses. Causes of oxidative stress include reactive oxygen species, ultraviolet rays, radiation, chemical substances, and stimuli that produce reactive oxygen species in cells. Oxidative stress is known to modify and damage lipids, proteins, and DNA in living organisms. When this degree is strong, it causes cell dysfunction and cell death, sometimes causing carcinogenesis, aging, arteriosclerosis, dementia, and neurological diseases. On the other hand, the existence of a mechanism for repairing this failure is also known. That is, in the living body subjected to oxidative stress, an antioxidant mechanism or an antioxidant substance is induced, and thus the damaged substance is repaired at the molecular level to protect the living body. Among these antioxidants, thioredoxin is one of the typical proteins responsible for the intracellular antioxidant mechanism.

  Thioredoxin was discovered in 1964 as a coenzyme for ribonucleotide reductase. This enzyme is a protein with a molecular weight of 12 kDa and has a -Cys-X-X-Cys- (Cys: cysteine, X: any amino acid) sequence well conserved between different species in the active site. Since this thioredoxin is induced by a cause or the like causing oxidative stress, the thioredoxin concentration reflects the degree of inflammation due to oxidative stress in the living body.

The currently reported method for measuring thioredoxin is an enzyme immunoassay (ELISA method) using an antigen-antibody reaction. For example, Non-Patent Document 1 shows that thioredoxin concentration in serum is useful as an index of oxidative stress in hepatitis C virus-infected patients using ELISA.
Yoshio Sumida, Toshiaki Nakashima, Takaharu Yoh, Yoshiki Nakajima, Hiroki Ishikawa, Hironori Mitsuyoshi, Yoshikuni Sakamoto, Takeshi Okanoue, Kei Kashima, Hajime Nakamura, Junji Yodoi Journal of Hepatology 2000, 33, 616-622.

  As disclosed in Non-Patent Document 1, the conventional method for measuring thioredoxin concentration uses an ELISA method. In this method, the target thioredoxin is recognized and captured by the antibody, and the amount of the captured thioredoxin is detected by using a label or the like. When the present inventors examined the ELISA method, it was found that there was a large variation in determination of the degree of stress, and there was room for further improvement as a stress sensor.

  Then, as a result of investigations on the cause of variation, when determining the degree of stress, it was better to determine whether the measured concentration of thioredoxins was an oxidant or a reductant. It was the first time that I was able to judge accurately. In the ELISA method, measurement is performed without distinguishing whether the thioredoxin in the measurement solution is an oxidized form or a reduced form. For this reason, in the conventional method for measuring thioredoxin, it is impossible in principle to measure the concentration of oxidized form or reduced form of thioredoxin or the ratio of oxidized form / reduced form.

  An object of the present invention is to provide an information acquisition device that distinguishes between an oxidized form and a reduced form of thioredoxin present in a sample solution and acquires information on the concentration thereof. Another object of the present invention is to provide an apparatus that uses this enzyme electrode to distinguish between oxidized and reduced forms of thioredoxin and to acquire information on these concentrations and concentration ratios. Another object of the present invention is to take advantage of the fact that the concentration ratio of oxidized form and reduced form of thioredoxin can be measured by such an information acquisition device, and the degree of stress from which information for classifying the degree of stress can be obtained. It is to provide a display device.

  The information acquisition apparatus of the present invention is an information acquisition apparatus for acquiring information relating to the concentration of thioredoxins, using the oxidation-reduction reaction of thioredoxins in a sample, It is an information acquisition device characterized by distinguishing and measuring at least one of the concentrations. Moreover, the stress sensor concerning this invention is a stress sensor which has the information acquisition apparatus of the said structure, and a stress degree determination part.

  The stress level information acquisition device of the present invention is a stress level information acquisition device for acquiring information on the stress level of a measurement target person, and utilizes a redox reaction of thioredoxins in a sample derived from the measurement target level. The concentration of the oxidized form of the thioredoxins and the concentration of the reduced form are separately measured, and the concentration is selected from the oxidized form concentration of the thioredoxins, the reduced form concentration, and the ratio of these concentrations. And a stress level determination means for determining the stress level of the person to be measured based on the second information related to the relationship between the first information and the stress level. It is.

  The stress level determination method of the present invention is a stress level determination method for determining the stress level of a measurement subject, and uses the oxidation-reduction reaction of thioredoxins in a sample from the measurement target to oxidize the thioredoxins. A stress level determination method comprising a step of determining a stress level based on at least one of a body concentration and a reductant concentration and a preset reference.

  The enzyme electrode of the present invention is an enzyme electrode having a conductive member and an enzyme, wherein the enzyme is an enzyme that catalyzes a redox reaction of thioredoxins.

  The method for measuring the concentration of thioredoxins according to the present invention is a method for measuring the concentration of thioredoxins in a sample, using the oxidoreductase reaction of thioredoxins in the sample, and the concentration and reduction of the oxidized form of thioredoxins. It is a method for measuring the concentration of thioredoxins, characterized by separately measuring at least one of the body concentrations.

  ADVANTAGE OF THE INVENTION According to this invention, regarding the density | concentration of thioredoxins, the information acquisition apparatus etc. which can distinguish whether it is the density | concentration of an oxidant, or the density | concentration of a reductant can be provided.

  The information acquisition apparatus for obtaining information relating to the concentration of thioredoxins of the present invention has a configuration for distinguishing and measuring at least one of the oxidized form concentration and the reduced form concentration of thioredoxins in a sample.

  In a preferred embodiment of such an apparatus, at least one of items selected from the concentration of oxidized form of thioredoxins, the concentration of reduced form, and the ratio of these concentrations is measured by utilizing the redox reaction of the thioredoxins. It has the composition of. Such an apparatus calculates one or more of the items based on a reaction region for reacting the sample with an enzyme that catalyzes a redox reaction of thioredoxins, and a reaction between the sample and the enzyme in the reaction region. And at least detecting means for

  The measurement of the concentration of thioredoxins and the acquisition of information related to the concentration in the present invention include the case of obtaining the concentration itself in the sample and the case of obtaining the absolute amount of the measurement object (compound) in the specified amount of sample. For example, taking 10 ml of blood and examining the absolute amount thereof is nothing but measuring the concentration in the present invention.

  First, thioredoxins will be described in detail. Thioredoxin has an oxidized form that forms a disulfide bond between the two cysteine residues described above and a reduced form that becomes dithiol. Of these, the reduced form functions as an antioxidant and eliminates reactive oxygen species by itself, and also eliminates reactive oxygen species in cells by the action of peroxiredoxin and becomes an oxidant. On the other hand, the oxidized form is converted into the reduced form by nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of thioredoxin reductase.

  The enzyme that catalyzes the redox reaction of thioredoxins distinguishes that the thioredoxins are oxidized or reduced, and catalyzes the reaction corresponding to the enzyme. For this reason, it is possible to measure the concentration of oxidized form of thioredoxins, the concentration of reduced form, or the concentration ratio of oxidized form and reduced form, which was impossible in principle by the conventional ELISA method.

  In this case, thioredoxin oxidase, thioredoxin dehydrogenase, thioredoxin reductase, and peroxiredoxin can be applied as the enzyme that catalyzes the oxidation-reduction reaction of thioredoxin. Among them, thioredoxin reductase is preferably used. This enzyme may be used alone or in combination with other enzymes that do not catalyze redox of thioredoxin. In particular, it is preferable to use an enzyme that catalyzes a reaction not involving thioredoxin in combination with an enzyme that catalyzes a redox reaction of thioredoxin. An example of this is the combination of thioredoxin reductase and ferredoxin NADP + reductase.

  The thioredoxins in this case include a series of proteins having a domain for thioredoxin called a thioredoxin superfamily in addition to thioredoxin (TRX). This thioredoxin superfamily molecule has an active site composed of -Cys-X-X-Cys-, and is a molecule that bears its redox ability by the disulfide / dithiol group of the active site. The thioredoxin superfamily also includes those having multiple thioredoxin motifs. Examples of these thioredoxin superfamily include TRX, Sptrx, PDI, ERp72 and ERdj5.

  The measurement method by enzyme reaction in this case is a measurement method in which an enzyme reaction is included in the reaction for measuring the concentration of thioredoxins. The concentration of thioredoxins is measured using at least one of an enzyme reaction that causes oxidoreductase to act on thioredoxins and an enzyme reaction linked to the redox reaction of thioredoxins. As described above, in addition to the reaction of thioredoxins with an oxidoreductase, another enzyme reaction for extracting a change detectable by the detection means may be linked to the oxidoreductase reaction of thioredoxins. The enzyme used for the enzyme reaction for concentration measurement can be used in a state of being immobilized on a carrier, that is, as an immobilized enzyme.

  The detection method for detecting the change obtained in the reaction region based on the enzyme reaction is not particularly limited. As a detection method, an enzyme electrode method, an absorbance method (including a colorimetric method), and a luminescence detection method are preferably used, and an enzyme electrode method and a colorimetric method are more preferably used.

Examples of this colorimetric method include a method using a change in absorbance at 340 nm when NADPH changes to NADP ⁺ , and a method using a dye in addition to NADPH. Examples of this dye include 5,5′-dithiobis (2-nitrobenzoic acid) and dithiothreitol.

  The environment for measurement in this case is not particularly limited, but an environment in which thioredoxins are present in a liquid is preferably used, and a solution containing water, alcohols, or an ionic liquid is more preferably used. It is done.

  The enzyme electrode includes at least an electroconductive member and an enzyme that catalyzes a redox reaction of thioredoxins. As the enzyme, if necessary, another enzyme may be used in combination with the enzyme that catalyzes the redox reaction of thioredoxins.

  The conductive member is used to extract the electrical changes generated by the enzyme reaction to an external circuit and measure it. It is highly conductive and has sufficient electrochemical stability under the conditions of the enzyme reaction. What consists of material which has is utilized. Examples of the constituent material of the conductive member include metals such as Au and Pt, conductive polymers such as polyacetylenes and polyarylenes, and metal oxides including In, Sn, and Zn. it can. Furthermore, examples of the constituent material of the conductive member include carbon materials such as graphite, carbon black, carbon nanotube, carbon nanohorn, and fullerene compound. Alternatively, these two or more composite materials, or a composite material in which a conductive material layer is provided on the surface of the substrate may be used.

  The immobilization of the enzyme to the conductive member is not particularly limited as long as the enzyme can be immobilized so that the target enzyme electrode function can be obtained, and a known method can be used. In addition to the enzyme, mediators such as metal complexes, quinones, and heterocyclic compounds that promote the transfer of electrons between the enzyme and the conductive member may be used in combination.

An information acquisition apparatus using the enzyme electrode method of thioredoxins can be constituted by the enzyme electrode having the above configuration. This information acquisition apparatus can be configured using at least the following components.
(1) A reaction region that can accommodate a sample solution.
(2) An enzyme electrode installed in the reaction region.
(3) Reaction detection means for measuring at least one of the above-mentioned items by detecting a reaction between the sample solution stored in the reaction region and the enzyme electrode as an electrical change.

  By using this apparatus, it is possible to measure at least one item of the concentration relating to the oxidized form and reduced form of thioredoxins, and the ratio relating to the concentration of oxidized form and reduced form.

  The enzyme electrode method is a measurement method in which an enzyme reaction is involved in the signal detected by the electrode reaction. Specifically, the concentration of the substance that reacts on the electrode changes due to the enzyme reaction, or between the electrodes. An example in which an electrical signal is changed by an enzyme reaction is mentioned. Examples of these include the former in which the concentration of redox substances including oxygen, hydrogen peroxide, quinones, and metal complex compounds on the electrode changes in relation to the enzyme reaction, and the latter between the electrodes. In which the impedance changes in relation to the enzyme reaction. Further, the distinction between the former and the latter is convenient, and it cannot be denied that a certain measurement method belongs to both. In this enzyme electrode, either or both of the enzyme and the substance that mediates the charge between the enzyme and the electrode may be immobilized on the electrode.

  When measuring using an electrode, it is preferable to increase the surface area by roughening the surface of the electrode or forming a material having a fine structure, and subjecting the surface of the electrode to a treatment that reduces the background current. . Examples of this treatment include a method of chemically modifying the electrode and a physical modification. As a specific example, the former includes adsorption of a thiol compound or a silane compound, and the latter includes covering the electrode surface with a fluororesin film or a dialysis membrane. Moreover, these methods may be used alone or in combination.

  By using an enzyme electrode, it is possible to calculate the concentration of at least one of oxidized form and reduced form of thioredoxin by measuring an electrochemical change based on an enzyme reaction involving at least one of oxidized form and reduced form of thioredoxin. . Examples of this electrical change include current, charge amount, potential, voltage, and impedance. One or more of these can be measured, and the concentration of at least one of the oxidized form and reduced form of thioredoxin can be calculated from the measured value. Can do. The current measured here may be a current resulting from a reaction of a substance on the electrode, and this may be a steady current or a transient current. The charge amount measured here may be an integration of observed current values, which may be total electrolysis of a specific substance in a sample solution or partial electrolysis. The potential measured here may be the potential of the active center of the enzyme or the potential of a compound whose oxidation-reduction ratio changes due to the enzyme reaction. It may be something like that. The impedance measured here may be one in which the impedance between the electrodes changes in relation to the enzyme reaction, and is evaluated using an imaginary component, a real component, or a combination thereof. Good.

  That is, when detecting an electrochemical change associated with an enzyme reaction, the electrochemical change is detected as at least one of current, charge amount, potential, voltage and impedance, and the desired concentration of oxidized or reduced form is detected. Can be calculated.

As a method for detecting an electrochemical change, the following specific examples can be further given.
(A) A method for evaluating the amount of electrons transferred in relation to an enzyme reaction that reduces an oxidized form of thioredoxins, using a current and a charge amount.
(B) A method for evaluating the amount of electrons transferred in association with an enzyme reaction that reduces an oxidized form of thioredoxins by using an electric current and a charge amount at an electrode using a mediator.
(C) A method in which the ratio of oxidized form / reduced form of a mediator that is oxidized in association with an enzymatic reaction that reduces oxidized form of thioredoxins is evaluated at the electrode by current, charge amount, potential, and voltage.

  In addition, said evaluation means calculating the density | concentration of a to-be-measured substance from the electric current, electric charge amount, electric potential, and voltage which were obtained by measurement according to the criteria provided beforehand, for example, a calibration curve.

  The source of thioredoxins to be measured is not particularly limited, but biological fluids and tissues are preferably used. Of these, animal body fluids are more preferably used, and human body fluids are most preferably used. The body fluid is not particularly limited, but blood, blood components (including serum and plasma), urine, and saliva are preferably used.

  In the apparatus in this case, separate processing is preferably performed before and after the measurement of thioredoxins. Examples of this treatment include separation, fractionation, filtration, washing, extraction, purification, temperature change, dispersion, mixing, precipitation, dialysis, distillation, modification (including chemical reactions), deoxygenation, defoaming, and sonication. , Microwave treatment, treatment with application of magnetic field, electrolysis, electrophoresis, chromatogram. One process selected from these can be used as necessary, or a combination of two or more processes can be used. A method of automating these pre- and post-processing and incorporating them as part of the apparatus is also preferably used. In addition, it is also preferable to use a minute space such as a microchannel for this pre- and post-processing and / or measurement.

  As a specific example of the measurement method using an enzyme reaction, there is a measurement method using a thioredoxin reductase (thioredoxin reductase). In this measurement method, thioredoxin reductase specifically acts on an oxidized form of thioredoxins contained in a sample, and is reduced to a reduced form. The oxidation-reduction reaction at that time can be detected by various detection methods, and the concentration of the oxidant can be determined separately from the reductant. Further, by using an oxidase of thioredoxins, the concentration of the reduced product in the sample can be obtained separately from the oxidized product.

  Furthermore, the concentration of the reduced form of thioredoxins in the sample can be determined using thioredoxin reductase. For example, the oxidant concentration in the sample is first measured, an oxidant that oxidizes thioredoxins is added to the sample to convert the reductant present in the sample into an oxidant, and the excess oxidant is converted by an enzymatic reaction or the like. Remove it. Next, a concentration measuring method similar to the method for measuring the concentration of oxidant in the sample is performed. And the density | concentration of a reductant is calculated | required by subtracting the oxidant density | concentration before oxidant addition from the detected numerical value.

The stress level information acquisition apparatus and the stress level determination method of the present invention determine the stress level of the measurement target person from the following first information and second information.
(1) First information obtained by distinguishing an oxidized form and a reduced form of thioredoxins in a sample from a measurement subject and measuring their concentrations.
(2) Second information relating to the relationship between the first information and the stress level.

  For the first information, at least one item of the oxidized form concentration of thioredoxin, the reduced form concentration, and the ratio of these concentrations can be used. This apparatus preferably includes a stress level determination means for classifying at least one of the above items relating to the concentration of thioredoxins based on the second information to determine the stress level of the measurement subject. The stress level determination unit of the stress sensor can be configured from the stress level determination means.

  An example of a block diagram of the constituent units of such an apparatus is shown in FIGS.

  The apparatus shown in FIG. 13 includes at least an input device, a CPU, and output means, and further includes a storage device and a display device provided as necessary. The concentration value of thioredoxins from the measurement subject or data indicating the concentration is input by the input device. Here, the data indicating the concentration is an absorbance or a current value corresponding to the detection method used for the concentration measurement. The CPU is written with a program for processing the input data based on preset criteria to classify and measure the stress level of the person being measured. The degree of stress determined by this program, that is, the determination result can be output by the output means, that is, the determination result output means. For example, when a display is used as the display means, the determination result is displayed on the display. Alternatively, the determination result may be output from an output unit to an appropriate medium such as paper.

  Pre-set criteria for classifying the stress level can be created based on statistically collected data. For example, the degree of stress is ranked in a plurality of stages, and the range of the concentration of oxidized form of thioredoxins corresponding to each rank is determined based on statistically collected data. The concentration of the oxidized form of thioredoxins in the sample from the measurement subject is classified according to the ranking, and the CPU automatically determines which rank of the stress the measurement subject has. As an index for classification and determination of the degree of stress, at least one item of the thioredoxin oxidant concentration, the reductant concentration, and the concentration ratio thereof can be used. Further, if necessary, the concentration of thioredoxins (oxidized body + reduced body) in the sample can be additionally used as an index.

  As shown in FIG. 13, by providing a storage device, it is possible to record data on the concentration of thioredoxins from the measurement subject and determination results, and for each hour, day, week, month or year It is also possible to cause the CPU to create data relating to changes over time.

  The apparatus shown in FIG. 14 further includes an information acquisition apparatus that measures the concentration of thioredoxins in a sample. As this information acquisition apparatus, it is preferable to use at least an apparatus that performs the measurement using the enzyme reaction described above.

As the measured value of the concentration of thioredoxins used in the stress degree information acquisition apparatus, a value obtained as follows can be used.
(1) Concentration of oxidized form of thioredoxins (1-1) Concentration of oxidized form of thioredoxins measured using a sample.
(1-2) A concentration obtained by subtracting the concentration of the reductant from the total concentration of thioredoxins as the sum of the oxidant and the reductant contained in the sample.
(1-3) The concentration of oxidant obtained from the total concentration of thioredoxins as the sum of oxidant and reductant contained in the sample and the concentration ratio of oxidant and reductant.
(2) Concentration of reduced form of thioredoxins (2-1) Concentration of reduced form of thioredoxins measured using a sample.
(2-2) A concentration obtained by subtracting the concentration of oxidant from the total concentration of thioredoxins as the sum of oxidant and reductant.
(2-3) The concentration of oxidant obtained from the total concentration of thioredoxins as the sum of oxidant and reductant contained in the sample, and the concentration ratio of oxidant and reductant.
(3) Concentration ratio of oxidized form and reduced form of thioredoxins (3-1) A ratio obtained from the concentration of oxidized form and reduced form measured using a sample.
(3-2) A ratio obtained from the total concentration of thioredoxins as the sum of the oxidized form and the reduced form and the concentration of the oxidized form.
(3-3) A ratio obtained from the total concentration of thioredoxins as the sum of the oxidant and the reductant and the concentration of the reductant.
(3-4) A ratio obtained by measuring the potential of the active site of thioredoxin or the potential of a substance whose potential changes in relation to the potential of the active site.

  Examples of a method for obtaining the ratio obtained by measuring the potential of the active site of thioredoxin or the potential of a substance whose potential changes in relation to the potential of the active site include the following methods.

First, in measuring the potential, there is an active site of thioredoxin and an electrode that performs electron transfer at a sufficient speed for measurement, or a substance that enables electrical connection between the active site and the electrode, and the active site of thioredoxin The measurement is performed under the condition that can measure the potential. Under this condition, the potential that changes depending on the ratio of oxidized form / reduced form of thioredoxin is calculated using the Nernst equation.
Nernst equation:
(E = E ₀ + (RT / nF) ln (a _O / a _R )
E: electrode potential E ₀ : standard electrode potential R: gas constant T: absolute temperature n: number of electrons involved in the reaction F: Faraday constant a _O : oxidant activity a _R : reductant activity thioredoxins As a method for measuring the total amount of the above, a method not using an enzyme may be combined. Examples of such a method include enzyme immunoassay (ELISA).

Thioredoxin, which is an antioxidant substance, is induced when oxidative stress is applied to a living body, and its concentration increases. Therefore, as in Non-Patent Document 1, it is used as an index of oxidative stress. But this indicator is
1. Generation of oxidative stress,
2. Detection of oxidative stress by living body,
3. 3. induction of thioredoxin, and It is indirect via multiple steps, such as increasing the concentration of thioredoxin.

  For this reason, there is a time difference between the occurrence of oxidative stress and the increase in the concentration of thioredoxin, and there is a difference in response between individuals in the course of going through these multiple steps, resulting in the observed thioredoxin concentration. There is a problem that individual differences occur. For this reason, it may be difficult to perform highly accurate stress evaluation, disease diagnosis, follow-up observation, and the like based on this index.

Therefore, the present inventors examined the relationship between the occurrence of oxidative stress and an index for evaluating it. In the process, attention was paid to the fact that the oxidized thioredoxin concentration (or oxidant / reductant ratio) in the living body responds in a smaller number of steps as compared with the thioredoxin concentration as shown in 1 to 4 below.
1. Occurrence of oxidative stress.
2. Elimination of oxidative stress by thioredoxin reductant and generation of thioredoxin oxidant.
3. Increased thioredoxin oxidant concentration (or oxidant / reductant ratio).
Furthermore, we focused on the fact that it does not include processes that are considered to have large individual differences such as detection of oxidative stress by the living body and induction of thioredoxin. Based on these points of interest, the inventors have reached a method for evaluating oxidative stress using the concentration of oxidized form of thioredoxin or the concentration ratio of oxidized form and reduced form as an index.

  In this method, as described above, the index is more direct to oxidative stress than the conventional method. For this reason, it is excellent in the time responsiveness from application of oxidative stress to detection. In addition, the oxidized form of thioredoxin is produced by oxidation of thioredoxin and directly reflects the applied oxidative stress. Therefore, the thioredoxin oxidized form has less individual differences than measuring the total amount of thioredoxin. . As a result, more accurate oxidative stress evaluation, disease diagnosis, follow-up observation, and the like can be performed.

  In addition, not only oxidative stress but also many stress response mechanisms have elements common to each other, and at the same time, various signal pathways are activated and closely related to perform regulation. Therefore, there is a possibility that other stresses can be evaluated by a technique that can evaluate oxidative stress according to the present invention.

As an index for stress evaluation in this case, the concentration of oxidized thioredoxins, or the concentration ratio of oxidized and reductant may be used alone or in combination, and other indicators and It is preferable to use in combination. The following methods can be given as examples of evaluation methods when used in combination.
(1) A method in which the stress level is classified and evaluated according to quadrants existing in the two axes x and y.
(2) A method of evaluating with the x and y coordinates of two axes and the area of a triangle having the origin as a vertex.
(3) A method of using a coordinate position on two axes as shown in FIG.
(4) A method of evaluating the x, y, z coordinates and the volume of a tetrahedron whose apex is the origin in the three axes x, y, z.
(5) A method for evaluating a pattern using the shape of a figure drawn in multiple axes as shown in FIG.

  In the case where the concentration ratio between the oxidized form and the reduced form of thioredoxin is used as a single index, the relationship between the concentration ratio and the degree of stress (stress level) is set in advance from statistically obtained data. For example, according to the concentration ratio, the stress level is set in four stages in descending order of the stress level from A to D. The concentration ratio of the oxidized form and the reduced form of thioredoxin contained in the sample derived from the measurement subject is measured by the above method, and the stress level of the measurement subject is classified into any one of A to D from the obtained measurement value. . This classification process can be automatically performed by computer processing the measured value of the concentration ratio of the oxidant and the reductant according to a predetermined program. In this case, the CPU of the computer constitutes the classification means. The classification result may be output by the output means via a desired medium such as paper or various displays, or may be stored and stored in the storage means so that it can be retrieved as necessary. Good. An example of a block diagram of the constituent units of such a stress level information acquisition apparatus is shown in FIG.

  In addition to the above classification means and classification result output means, the stress level information acquisition apparatus of the present invention is for measuring at least one of the thioredoxin oxidant concentration, the reductant concentration, and the concentration ratio thereof. It may have an information acquisition device. FIG. 14 shows a block diagram of an example of a stress degree information acquisition device to which a concentration information acquisition device is added.

  The stress level information acquisition apparatus and stress level determination method of the present invention can be suitably used for acquiring information related to a stress level that is useful for diagnosing a disease and observing the progress of the disease. The disease is not particularly limited as long as it is accompanied by a change in the concentration related to the oxidized form of thioredoxins and the ratio related to the oxidized form and reduced form concentration of thioredoxins. Examples of this disease include lung disease, cardiovascular disease, liver disease, gastrointestinal disease, kidney disease, diabetes, acquired immune deficiency syndrome, tumor, skin disease.

  The stress level information acquisition apparatus and stress level determination method of the present invention are used for evaluating or predicting the effect of thioredoxins before administration in the case of using thioredoxins as a medicament, and for evaluating or predicting the effect after administration. Therefore, it can be suitably used.

  For example, the possibility of a disease may be displayed on the display device based on the stress level of the measurement subject determined by the stress level information acquisition apparatus in the apparatus shown in FIGS. In this case, a program for selecting a possible disease according to the degree of stress determined in advance based on data obtained by statistically collecting the correlation between the degree of stress and the disease. Such processing can be performed by incorporating it in the CPU. In addition, the time-dependent data of the measurement subject is stored in the storage device, and the change in the stress level of the measurement subject over time can be output, and the progress of the related disease is measured for each measurement based on this change. It is also possible to classify and display such as “good progress” or “no change”. Such classifications can also be created according to criteria created based on statistically collected data.

  The stress level information acquisition apparatus and stress level determination method of the present invention are used for evaluating or predicting the effect of thioredoxins before administration in the case of using thioredoxins as a medicament, and for evaluating or predicting the effect after administration. Therefore, it can be suitably used.

  For example, the memory device of the apparatus shown in FIG. 13 and FIG. 14 stores a pattern of stress level at which medication is effective, and compares this pattern with data from the measurement subject to administer medication for the measurement subject. It is also possible to create data that assists in predicting the effectiveness of.

  In addition, the stress level change pattern (for example, change over time) when the medication is effective or not effective is stored in the apparatus of FIGS. 13 and 14 as saved data. Then, using this stored data, the evaluation of whether the medication was effective or could be predicted to be effective by comparing the data from the measurement subject who performed the medication with the pattern of the change in the stress level. Can also be done. A stress level pattern and a stress level change pattern used as a basis for these evaluations can also be created based on statistically collected data.

  The present invention will be described in more detail with reference to the following examples. However, the method of the present invention is not limited to these examples.

Example 1
NADPH Enzyme Electrode System FIG. 3 is a diagram showing an example of an apparatus for measuring the concentration related to the oxidized form or reduced form of the thioredoxins of the present invention or the concentration ratio of oxidized form and reduced form. The portion shown as the upper surface in FIG. 3 shows a plan view obtained by developing each member in order from the top to the bottom. 3 is a cross-sectional view in the vertical direction of the apparatus. FIG. 3 shows an example of the basic structure of the sensitive part of the thioredoxin oxidant concentration sensor using an enzyme electrode.

  The sensitive part in this apparatus mainly comprises a reaction tank cover 1, a reaction tank wall 2, a substrate 3, and an insulating layer 4. The reaction vessel cover 1 is made of, for example, polyethylene terephthalate (PET) resin with an adhesive, and provided with openings 5 serving as sample introduction ports and air discharge ports, which are respectively positioned diagonally to the reaction vessel 6. is doing. The reaction vessel wall 2 is made of, for example, polydimethylsiloxane (PDMS), and is designed to hold a certain amount of sample (500 μL, for example) on the electrodes 7, 8, 9 of the substrate 3. For the substrate 3, for example, a plate material made of polyimide is used, and a working electrode 8, a counter electrode 9, and a reference electrode 7 are provided on the substrate 3. Each electrode is connected to the back surface of the substrate 3 through the through hole 13 and is connected to the current collecting pad 12 through the lead 11. The through hole 13 and the lead 11 are previously formed by plating a copper-clad polyimide substrate or photolithography.

  As the working electrode 8, a glassy carbon electrode treated with polyaminoaniline is used. A thin piece is cut out from the glassy carbon rod and bonded to a temporary substrate (not shown) with a conductive paste. A glassy carbon cylinder that becomes the working electrode 8 in a 1.0 M sulfuric acid aqueous solution containing 0.01 M 2-nitroaniline using a lead from the back and a reference electrode and a counter electrode (not shown) for polyaminoaniline treatment A potential is applied to the thin slice using a potentiostat. A cycle of 1.5 V for 10 seconds and -0.5 V for 50 seconds was repeated for 1 hour, then -0.5 V was applied for 10 minutes, and then washed with water to obtain an electrode serving as a working electrode. After washing with water, this electrode is removed from the temporary substrate and bonded to the sensor substrate 3 with a conductive paste. The counter electrode 9 is prepared by sputtering Ti / Pt on the substrate 3. As an example, the film thickness is Ti 100 nm and Pt 200 nm. The reference electrode 7 is prepared by forming Ti / Pt similarly to the counter electrode 9, further forming an Ag layer by sputtering, and then performing chlorination treatment. An example of the thickness of the Ag layer is 500 nm. The thickness of the Ag layer needs to be optimized depending on the usage environment and time. The chlorination treatment of the Ag layer is performed by immersing in a 50 mM aqueous solution of FeCl3 for 10 minutes. On each electrode, a fixed composition and a fixed amount of reagent layer are applied. The reagent layer is prepared in advance so that it can be easily dissolved in an aqueous solution by mixing a certain amount of enzyme, if necessary, an enzyme carrier and a mediator molecule. The reagent layer preparation method will be described below.

  An aqueous solution is prepared by mixing 50 nmol of a viologen derivative immobilized on alginic acid (corresponding to a viologen molecule), NADPH 1 μmol, ferredoxin NADP + reductase 0.1 unit, and thioredoxin reductase 1 unit. This aqueous solution is dropped on the working electrode 8 and then dried in a drying tank.

  Next, a method for synthesizing the viologen derivative immobilized in the alginate layer will be described.

  An equimolar amount of methyl iodide and 1,4-dibromobutane are added to 4,4′-bipyridine, and the mixture is reacted at 110 ° C. for 6 hours in an autoclave. The raw material was removed from the obtained product by distillation under reduced pressure, the water-soluble component was applied to a silica gel column, and the desired 1-methyl-1'-bromobutyl-4,4'-bipyridine bromide, iodide salt (BrBuV) was added. obtain. Commercially available sodium alginate (molecular weight 20000) 28.7 mg and 0.15 mmol of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) are dissolved in 20 mL of water. After stirring for 40 minutes, 0.75 mmol of commercially available polyethylene oxide diamine is added and further stirred for 1 hour. This solution is dialyzed against water for 24 hours, 0.3 mmol of BrBuV is added, and dialyzed against water at 5 ° C. for 12 hours to obtain a viologen derivative immobilized on alginic acid.

  Prior to the measurement, oxidized and reduced thioredoxins are prepared. The oxidized form of thioredoxin can be separated by gel filtration chromatography after adding a sufficient amount of hydrogen peroxide to a phosphate buffer containing a commercially available thioredoxin and a sufficient amount of peroxiredoxin to oxidize the thioredoxin. get. On the other hand, a reduced form of thioredoxin is obtained by adding a sufficient amount of NADPH to a phosphate buffer solution containing a sufficient amount of thioredoxin reductase to reduce the thioredoxin, and then separating and obtaining it by gel filtration chromatography.

Connect the measuring part to a potentiostat, and prepare a solution of oxidized thioredoxin in 50 mM phosphate buffer pH 7.0, and a solution of reduced thioredoxin in 50 mM phosphate buffer pH 7.0. . After adjusting the temperature to 37 ° C. and bubbling with nitrogen, the solution prepared from the injection port of the measurement unit is added, and a potential of −0.9 V is applied to the working electrode with respect to the reference electrode. When the observed steady-state current amount or accumulated charge amount is plotted on the vertical axis and the thioredoxin concentration of the added oxidant or reductant is plotted on the horizontal axis, the observed charge amount has a tendency as shown in FIG. That is, in a sample in which an oxidized form of thioredoxin is added to the buffer, the steady-state current value or the amount of charge increases as the concentration of thioredoxin increases. In contrast, in a sample in which a reduced form of thioredoxin is added to the buffer, the steady-state current value or the amount of charge does not increase even when the concentration of thioredoxin is increased. In short, in the case of an oxidized form, the oxidized form of thioredoxin receives electrons from NADPH through the catalytic action of thioredoxin reductase, and the reaction shown in FIG. This reaction amount is proportional to the amount of oxidized thioredoxin in the solution, that is, the amount of thioredoxin that can be reduced in the solution. On the other hand, in the case of a reduced form, thioredoxin is already a reduced form and cannot receive electrons from NADPH. In addition, when quantifying the amount of thioredoxin reductant (R / mol) using this measurement unit, as a pretreatment in advance, the sample solution is expected to have a certain amount of peroxiredoxin than the expected amount of thioredoxin reductant. A large molar amount of hydrogen peroxide is added. Thereby, the reduced form of thioredoxin in the solution is set as an oxidant. Thereafter, catalase is added to the system to decompose excess H ₂ O ₂ . Then, the charge amount (X / C) is measured by the same method as that for measuring the amount of oxidized thioredoxin. Then, using the oxidant amount of thioredoxin in solution (O / mol) and X, the amount of reductant in the sample using the formula (R) = (X / 2F)-(O) (F: Faraday constant) Can be requested. In this case, the graph in the case where the amount of reductant calculated on the vertical axis and the thioredoxin concentration of each of the oxidant and reductant solutions on the horizontal axis has a tendency as shown in FIG.

Example 2
NADPH colorimetric system FIG. 7 shows an apparatus for measuring the concentration of oxidized form or reduced form of the thioredoxins of the present invention, or the concentration ratio of oxidized form and reduced form, specifically, oxidation of thioredoxin using a colorimetric method. It is a figure for demonstrating the basic process of a body and a reductant concentration sensor. This sensor has a cell constituting a reaction region, and a detection means having a light source and a light receiving element for measuring an optical change based on a reaction occurring in the cell. The measurement process using this sensor mainly includes an optical cell introduction process, a sample introduction process, a reaction process, a detection process, and a discharge process. In the optical cell introduction step, the optical cell into which the reaction reagent has been introduced is fixed to the holder. An example of the reaction reagent is a phosphate buffer in which thioredoxin reductase and NADPH are dissolved. As an example of the concentration and amount, 0.5 mL of 0.1 M phosphate buffer pH 7.0 containing 0.1 unit of thioredoxin and 50 nmol of NADPH can be mentioned. In the sample introduction process, a sample is introduced into the optical cell. An example of the amount of the sample introduced is 0.5 mL. In the reaction step, the solution is stirred and the cell is maintained at a temperature suitable for the enzymatic reaction (eg, 25 ° C.). The reaction time at this time may take a sufficient time (for example, 20 minutes) for all the oxidized form of thioredoxin to be converted into a reduced form, or the time after introduction of the sample is accurately measured and changed. You may observe the light absorbency in. In the detection step, light obtained by separating the monochromatic light source, multicolor light source, and multicolor light source with a grating or the like is transmitted through the optical cell, and the transmitted light is detected by the light receiving element. In the discharging process, the optical cell whose measurement has been completed is discharged.

As a reaction reagent, 0.5 mL of 0.1 M phosphate buffer pH 7.0 containing 0.1 unit of thioredoxin reductase and 50 nmol of NADPH is used. As a sample, 0.5 mL of 0.1 M phosphate buffer pH 7.0 containing 0 to 10 nmol of oxidized or reduced thioredoxin is used. For each sample, the absorbance observed at the detection site when a reaction time of 25 minutes at 25 ° C. is applied. On the other hand, 0.5 mL of a phosphate buffer solution containing no thioredoxin is used as a comparison target of this sample, and the absorbance observed at the detection site when a reaction time of 20 minutes is applied at 25 ° C. is obtained. With respect to the obtained absorbance, the value of (Comparison Object)-(Sample) is plotted on the vertical axis, and the concentration of added thioredoxin is plotted on the horizontal axis, and there is a tendency as shown in FIG. Here, NADPH has an absorption coefficient of 6220 M ⁻¹ cm ⁻¹ at 340 nm, whereas this oxidant, NADP ⁺ has no absorption. In the sample in which an oxidized form of thioredoxin is added to the buffer, the absorbance at 340 nm decreases with respect to the comparison object as NADPH changes to NADP ^{+ as} the concentration of thioredoxin increases. On the other hand, in the sample in which the reduced form of thioredoxin was added to the buffer, increasing the concentration of thioredoxin did not change the NADPH concentration and the absorbance. In short, in the case of a solution to which an oxidized form of thioredoxin is added, the oxidized form of thioredoxin receives electrons from NADPH through the catalytic action of thioredoxin reductase, and the reaction shown in FIG. This amount of reaction is proportional to the amount of oxidized form of thioredoxin in solution, that is, the amount of thioredoxin that can be reduced in solution. On the other hand, in the case of a reduced form, thioredoxin is already a reduced form and cannot receive electrons from NADPH.

  In addition, when quantifying the amount of reduced thioredoxin (R / mol) using this sensor, as a pretreatment, a certain amount of peroxiredoxin and hydrogen peroxide are added to the sample solution in advance, and thioredoxin in the solution is added. The reductant is left as an oxidant. Thereafter, the absorbance is measured by the same method as that for measuring the amount of oxidized thioredoxin, and the difference (Y) from the comparison target is calculated. Then, using the amount of hydrogen peroxide added (H / mol), the amount of oxidized thioredoxin in solution (O / mol) and the amount of NADPH corresponding to Y (Y '/ mol), (R) = (H) It can be calculated by the equation + (O)-(Y ′). In this case, the graph in the case where the amount of reductant calculated on the vertical axis and the thioredoxin concentration of each of the oxidant and reductant solutions on the horizontal axis has a tendency as shown in FIG.

Example 3
Preprocessing
Blood is collected from the upper arm of hepatitis C patients and healthy subjects, and centrifuged at 3000 rpm for 10 minutes to obtain serum. The thioredoxin oxidant and reductant concentrations in the serum are measured using the electrode of Example 1. On the other hand, the thioredoxin concentration in the same serum is measured by a commercially available sandwich ELISA method (kit manufactured by Redox Bioscience). The measurement result at this time has a tendency as shown in FIG. That is, when the oxidant concentration of thioredoxin is used as an index, the individual difference is smaller than when the concentration of thioredoxin as a whole is used as an index, and the responsiveness to oxidative stress is improved. This is because the relationship between the measured thioredoxin concentration and oxidative stress is: 1. Oxidative stress generation, 2. Detection of oxidative stress by the living body, 3. Induction of thioredoxin, 4. Increased thioredoxin concentration, Indirect through multiple steps. On the other hand, the oxidant concentration of thioredoxin directly increases as thioredoxin undergoes oxidation, and directly reflects the applied oxidative stress. For this reason, it is considered that there is less individual difference than measuring the total amount of thioredoxin, and it becomes possible to evaluate responsive oxidative stress.

Example 4
Stress Evaluation FIG. 12 shows, as an example of evaluation of oxidative stress, the vertical axis represents the total concentration of thioredoxin and the horizontal axis represents the ratio of oxidized form / reduced form of thioredoxin. Here, it is considered that a person belonging to the region indicated by the symbol A may be subjected to strong oxidative stress, and if this is caused by a disease, the symptoms may be improved by administration of thioredoxin as a medicine It can be considered that there is sex. Next, the person belonging to the region indicated by symbol B has a high thioredoxin concentration, but did not receive strong oxidative stress, or was once subjected to strong oxidative stress, but may have decreased now Can be considered. In addition, even if a person belonging to the region indicated by the symbol B has a disease that causes oxidative stress, it can be considered that there is no high possibility that the symptoms are improved by administration of thioredoxin as a pharmaceutical. This is because, in the situation where a large amount of reduced thioredoxin already exists in the body, even if thioredoxin is administered from the outside, the possibility that oxidative stress is reduced is not so high. Next, it can be considered that a person belonging to the region indicated by the symbol C is highly likely not to receive strong oxidative stress from either index. Finally, it is highly likely that a person belonging to the region indicated by the symbol D is highly likely not to be subjected to strong oxidative stress only by determining the concentration of the entire thioredoxin. However, it may have been subjected to strong oxidative stress recently, that is, it may be in a transitional period until thioredoxin is induced and the total concentration of thioredoxin is increased, and it may be a constitution that has low ability to induce thioredoxin Can think. When a person belonging to the region indicated by the symbol D has a disease that causes oxidative stress, it can be considered that there is a high possibility that the symptom is improved by administration of thioredoxin as a medicine.

  Thus, compared with the case where the total concentration of thioredoxin is used as an index, the oxidized / reduced substance ratio of thioredoxin is introduced as a further index. As a result, it is possible to obtain new and useful findings such as the influence on the living body due to the current oxidative stress, the time when the oxidative stress is applied, and the effectiveness when thioredoxin is used as a medicine.

Example 5
Enzyme electrode reductant measurement system FIG. 15 is a diagram showing an example of an apparatus for measuring the concentration of the thioredoxins of the present invention relating to the reductant and oxidant, or the concentration ratio of oxidant and reductant. The basic structure of the measurement electrode site is the same as that shown in FIG. 3 except that the method for preparing the working electrode 8 is different, the reagent layer 10 is not provided, and the enzyme layer 14 is provided. The preparation method will be described below focusing on this difference.

  The working electrode 8 is composed of, for example, a polymer containing an osmium complex on a carbon electrode and an enzyme immobilized thereon. This preparation method will be described below.

A commercially available carbon paste screen printing method is applied onto the substrate 3, and hydrophilicity is achieved by UV-O ₃ treatment. A working electrode is prepared by dropping a mixture of an osmium complex-containing polymer, an enzyme, and an aqueous crosslinking agent solution (50 μLcm ⁻² as an example of a dripping amount) and drying it. Examples of dripping liquid, Compound 1 having the following structure, horseradish peroxidase, consist of polyethylene glycol diglycidyl ether, examples of the concentration, respectively 5mgmL ^-1, 1mgmL ^-1, include 0.2MgmL ^-1 .

  A method for preparing Compound 1 will be described.

20 mL of ethylene glycol, 0.08 g of (NH ₄ ) ₂ [OsCl ₆ ], and 0.38 g of 4,4′-dimethyl-2,2′-bipyridine were added to a 100 mL eggplant flask equipped with a reflux tube. Next, 300 W was irradiated for 20 minutes with a stirrer and a microwave synthesizer (Milestone microsynth) under a nitrogen stream. After the solution was cooled to room temperature, 25 mL of water in which 0.4 g of Na ₂ S ₂ O ₄ was dissolved was added. The black purple precipitate produced after stirring at room temperature for 1 hour was filtered and washed with water to remove excess salt. Thereafter, washing with diethyl ether, except for the ligand of unreacted under reduced pressure, drying by heating to 60 ° C., the Os (4,4'-dimethyl-2,2'- bipyridine) 2 Cl 2 Obtained.

To a 100 mL three-necked flask equipped with a thermometer and a reflux tube, 15 mL of water, 2.63 g of acrylamide, 0.403 mL of 1-vinylimidazole, 0.069 mL of N, N, N ′, N′-tetramethylethylenediamine were added. . Under a nitrogen stream, 0.06 g of ammonium persulfate was further added. The mixture was heated in a water bath at 40 ° C. for 30 minutes, and then the reaction vessel was air-cooled. The resulting viscous liquid is dropped into 500 mL of methanol with strong stirring and allowed to settle, and the precipitate is collected by centrifugation, dissolved by adding the minimum amount of water that can dissolve the precipitate, and further with 500 mL of strongly stirring. This aqueous solution was dropped into methanol and allowed to settle. The precipitate was collected again by centrifugation, and dried by heating to 60 ° C. under reduced pressure to obtain a polyacrylamide-polyvinylimidazole 7.49 / 1 copolymer. The formation of molecules and the unit ratio were determined by ¹ HNMR measurement (D ₂ O).

A 100 mL eggplant flask equipped with a reflux tube was prepared. To this, 25 mL of ethylene glycol, 17.5 mL of ethanol, 0.19 g of Os (4,4′-dimethyl-2,2′-bipyridine) ₂ Cl ₂ prepared previously, 0.22 g of polyacrylamide-polyvinylimidazole The copolymer was added. Next, 400 W was irradiated for 2 hours with a stirrer stirring and a microwave synthesizer under a nitrogen stream. After the solution was cooled to room temperature, 20 mL of ethanol was further added to the viscous precipitate produced by dropping the solution added with 20 mL of ethanol into 500 mL of diethyl ether solution with strong stirring. This was again added dropwise to 500 mL of diethyl ether solution under strong stirring, and the resulting viscous precipitate was dried by heating to 60 ° C. under reduced pressure. To 0.0755 g of the dried complex polymer, 0.59 g of hydrazine and 1.6 mL of water were added, heated at 40 ° C. for 6 hours, 150 mL of ethanol was added, and 90% of the solvent was removed by distillation under reduced pressure. Then, the complex polymer described in the target formula (1) was obtained by centrifuging and recovering the precipitate formed by dropping the supernatant into ether and drying under reduced pressure.

As the enzyme layer 14, for example, a polyvinylidene fluoride film in which peroxiredoxin is retained is used. An example of the holding amount is 250 unit cm ⁻² . This can be prepared by dropping an aqueous enzyme solution onto the membrane and drying it. This film is disposed so as to cover the working electrode 8.

Connect the measuring part to a potentiostat, and prepare a solution of oxidized thioredoxin in 50 mM phosphate buffer pH 7.0, and a solution of reduced thioredoxin in 50 mM phosphate buffer pH 7.0. . After adjusting the temperature to 37 ° C and bubbling with nitrogen, add the solution prepared from the injection port of the measurement unit, and add a hydrogen peroxide solution with a molar amount of 1 to several times the expected molar amount of thioredoxin. To do. A potential of +0.2 V is applied to the working electrode with respect to the reference electrode. When the observed steady-state current amount or the accumulated charge amount is plotted on the vertical axis, and the thioredoxin concentration of the added oxidant or reductant is plotted on the horizontal axis, the observed charge amount has a tendency as shown in FIG. That is, in a sample in which a reduced form of thioredoxin is added to the buffer, the steady-state current value or the amount of charge decreases as the thioredoxin concentration increases. In contrast, in a sample in which an oxidized form of thioredoxin is added to the buffer, the steady-state current value or the amount of charge does not decrease even when the concentration of thioredoxin is increased. This meaning can be explained with reference to FIG. In the absence of a reduced form of thioredoxin, the oxidation reaction of H ₂ O _{2 in} solution can be observed as current and charge on the electrode by the catalytic reaction of horseradish peroxidase and the electron transfer action of the osmium complex. Here, when a reduced form of thioredoxin is present in the system, a reaction in which H ₂ O ₂ in the solution is consumed by the reduced form of thioredoxin by the catalytic action of peroxiredoxin in the enzyme layer. For this reason, when a reduced form of thioredoxin is present, the current and charge resulting from the oxidation reaction of H ₂ O ₂ are reduced, and this reduction amount is the amount of reduced form of thioredoxin in the solution, that is, oxidized in the solution. It is proportional to the amount of thioredoxin that can be made. In contrast, in the case of an oxidized form, since thioredoxin is already an oxidized form, it does not oxidize any more even if horseradish peroxidase is present. Therefore, even if the concentration of oxidized form of thioredoxin is increased. The steady current value or the charge amount does not decrease.

In addition, when the amount of oxidized thioredoxin (O / mol) is quantified using this measurement part, the amount of thioredoxin reductase in the sample solution is expected to be larger than the expected amount of thioredoxin oxidized in the sample solution beforehand. A molar amount of NADPH is added. Thereby, the oxidized form of thioredoxin in the solution is set as a reduced form. Thereafter, NADPH oxidase and oxygen are introduced into the system, and surplus NADPH is used as NADP ⁺ . Then, the amount of charge (X / C) is measured by the same method as that for measuring the amount of reduced form of thioredoxin. The amount of thioredoxin in the solution (R / mol) and X are used to calculate the oxidant in the sample using the formula (O) = (X / 2F)-(R) (F: Faraday constant). The amount can be determined. In this case, the graph in the case where the amount of reductant calculated on the vertical axis and the thioredoxin concentration of each of the oxidant and reductant solutions on the horizontal axis has a tendency as shown in FIG.

FIG. 19 shows an apparatus for measuring the concentration of oxidized form or reduced form of the thioredoxins of the present invention, or the concentration ratio of oxidized form and reduced form, specifically, oxidized form of thioredoxin using a modified electrode. It is a figure for demonstrating the basic process of a reductant concentration sensor. This sensor has a cell that constitutes a reaction region, and detection means having an electrochemical measurement cell for measuring an electrochemical change based on a reaction that occurs in the cell. The measurement process using this sensor mainly includes a sample introduction process, a drug introduction process, a reaction process, a reaction solution introduction process, a detection process, and a discharge process. In the sample introduction step, a certain amount of measurement sample is introduced into the reaction vessel. In the drug introduction step, a drug used for the reaction is introduced into the reaction container. An example of the reaction reagent is a phosphate buffer in which thioredoxin reductase and NADPH are dissolved. In the reaction step, in the reaction step, the solution is stirred, the cell is maintained at a temperature suitable for the enzyme reaction, and the sample and the added drug are reacted. The reaction time at this time may take a sufficient time (for example, 20 minutes) for all the oxidized form of thioredoxin to be converted into a reduced form, or the time after introduction of the sample is accurately measured and changed. The signal inside may be observed in a later detection step. In the subsequent reaction solution introduction step, when the electrochemical cell does not serve as the reaction cell, the solution after the reaction is transferred to the electrochemical cell. In the detection step, a potential is applied from the potentiostat to the gold electrode, and response current and charge are detected and recorded. Thereafter, in the discharging step, the measured reaction solution is discharged.

  FIG. 20 is a diagram showing an example of an apparatus for measuring the concentration of the thioredoxins of the present invention relating to the reduced form and oxidized form, or the concentration ratio of the oxidized form and reduced form. The basic structure of the measurement electrode part is the same as that shown in FIG. 3, except that the method for preparing the working electrode 8 is different and the reagent layer 10 is not provided. The preparation method will be described below focusing on this difference.

  The working electrode 8 is made of, for example, a gold electrode and a molecule modified on the gold electrode, for example, pyrroloquinoline quinone (PQQ). This preparation method will be described below.

Gold is deposited on the substrate 3 by vapor deposition, sputtering or the like using titanium as a base. The gold electrode is cleaned by UV-O ₃ treatment. Drip cystameine aqueous solution on it and wash with water. Thereafter, a buffer solution of PQQ containing N- (3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (EDC), which is a coupling material, is dropped and washed with the buffer solution. As a drug, 0.5 mL of 0.1 M phosphate buffer pH 7.0 containing 0.1 unit of thioredoxin reductase and 50 nmol of NADPH is used. As samples, 0.5 mL of 0.1 M phosphate buffer pH 7.0 containing 0 to 10 nmol of oxidized or reduced form of thioredoxin is used. For each sample, the current value or charge amount observed at the detection site when a reaction time of 25 minutes at 25 ° C. is applied is measured. On the other hand, using 0.5 mL of a phosphate buffer solution containing no thioredoxin as a comparison target of this sample, the current value or charge amount observed at the detection site when a reaction time of 20 minutes is applied at 25 ° C. is obtained.

  Connect the electrode part to a potentiostat, adjust the reaction solution to 37 ° C, bubbling nitrogen, add the solution prepared from the injection port of the measurement part, and apply a potential of 0.2 V to the working electrode. . When the observed steady-state current amount or accumulated charge amount is plotted on the vertical axis, and the thioredoxin concentration of the added oxidant or reductant is plotted on the horizontal axis, the observed charge amount has a tendency as shown in FIG.

  In the sample in which an oxidized form of thioredoxin is added to the buffer, the steady-state current value or the amount of charge decreases as the thioredoxin concentration increases. On the other hand, in a sample in which a reduced form of thioredoxin is added to the buffer, even if the concentration of thioredoxin is increased, the steady-state current value or the amount of charge does not decrease. This phenomenon will be described with reference to FIG. At the electrode, a current due to the oxidation reaction of NADPH through PQQ is observed, and this current value or charge amount increases or decreases in proportion to the concentration of NADPH in the solution. In addition, when thioredoxin is an oxidant, it is reduced by NADPH due to the catalytic action of thioredoxin reductase, and the concentration of NADPH in the solution decreases. On the other hand, when thioredoxin is a reduced form, the concentration of NADPH in the solution does not decrease because it can no longer be reduced by NADPH.

  In addition, when the amount of reduced form (R / mol) of thioredoxin is quantified using this sensor, as described in Example 2, there is a method in which a reduced form of thioredoxin in a solution is used as an oxidant as a pretreatment. Available.

  ADVANTAGE OF THE INVENTION According to this invention, the apparatus which measures the density | concentration regarding the oxidant of a thioredoxin, a reductant, or the ratio regarding an oxidant and a reductant concentration can be provided, and it can utilize as an apparatus which evaluates the stress using these as an index It is.

This is an example of an evaluation method using two types of indices for stress evaluation and using a coordinate position on two axes. It is an example of a method for evaluating a pattern using a shape of a figure drawn in multiple axes using a plurality of indices as stress evaluation. It is the schematic of the oxidant concentration sensor sensitive part of the thioredoxin using an enzyme electrode. It is the conceptual diagram of the thioredoxin density | concentration dependence of the electric current measured using the enzyme electrode, or an accumulated charge amount. It is a schematic diagram of a series of reaction regarding the reduction | restoration of the thioredoxin oxidation body which advances with an enzyme electrode. It is a conceptual diagram of the added thioredoxin concentration dependence of the amount of reduced forms of thioredoxin measured using an enzyme electrode. It is a conceptual diagram of an oxidized form and reduced form concentration sensor of thioredoxin using a colorimetric method. It is a conceptual diagram of the thioredoxin density | concentration dependence of the light absorbency change from the comparison object of the thioredoxin using a colorimetric method. It is a schematic diagram of the reduction | restoration reaction of the thioredoxin oxidation body which advances at the reaction process. It is a conceptual diagram of the thioredoxin concentration dependence of the amount of reduced forms of thioredoxin measured using a colorimetric method. It is the conceptual diagram of the result of having performed the stress evaluation of the hepatitis C patient and a healthy subject using the oxidant concentration of thioredoxin or the density | concentration of the whole thioredoxin as a parameter | index. It is a conceptual diagram of the biaxial evaluation when using two types of indices of the concentration of thioredoxin as a whole and the thioredoxin oxidant / reducer ratio as stress evaluation. It is a block diagram which shows an example of a structure of a stress level information acquisition apparatus. It is a block diagram which shows an example of a structure of a stress level information acquisition apparatus. It is the schematic of the reduced body concentration sensor sensitive part of the thioredoxin using an enzyme electrode. It is the conceptual diagram of the thioredoxin density | concentration dependence of the electric current measured using the enzyme electrode, or an accumulated charge amount. It is a schematic diagram of a series of reactions relating to oxidation of a thioredoxin reductant proceeding at an enzyme electrode. It is a conceptual diagram of the added thioredoxin concentration dependence of the amount of thioredoxin measured using the enzyme electrode. It is a conceptual diagram of a thioredoxin oxidant and reductant concentration sensor using a NADPH modified electrode system. It is the schematic of the reduced body concentration sensor sensitive part of the thioredoxin using a modified electrode. It is the conceptual diagram of the concentration dependence of the oxidant and the reductant of the thioredoxin of the electric current and electric charge amount which used the NADPH modification electrode system. It is a schematic diagram of a series of reactions related to oxidation of a thioredoxin reductant proceeding at a NADPH modified electrode.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Reaction tank cover 2 Reaction tank wall 3 Substrate 4 Insulating layer 5 Sample introduction port, air discharge port 6 Reaction tank 7 Reference electrode 8 Working electrode 9 Counter electrode 10 Reagent layer 11 Lead 12 Current collecting pad 13 Through hole 14 Enzyme layer

Claims (19)

An information acquisition device for acquiring information on the concentration of thioredoxins,
An information acquisition apparatus characterized by distinguishing and measuring at least one of an oxidized form concentration and a reduced form concentration of a thioredoxin using a redox reaction of a thioredoxin in a sample. In order to measure at least one of the items selected from the oxidant concentration, the reductant concentration, and the ratio of these concentrations of the thioredoxins,
A reaction region for reacting the sample with an enzyme that catalyzes a redox reaction of thioredoxins;
Detection means for calculating one or more of the items based on a reaction between the sample and the enzyme in the reaction region;
The information acquisition apparatus according to claim 1, comprising:   The information acquisition apparatus according to claim 1, wherein the enzyme is a thioredoxin reductase.   The information acquisition apparatus according to claim 1, wherein the enzyme is an immobilized enzyme immobilized on a carrier, and the immobilized enzyme is disposed in the reaction region.   The information acquisition apparatus according to claim 1, wherein the detection unit detects an optical change based on the reaction and calculates one or more of the items.   The information acquisition apparatus according to claim 1, wherein the detection unit detects an electrochemical change based on the reaction and calculates one or more of the items.   The information acquisition apparatus according to claim 6, wherein the detection unit includes an enzyme electrode.   The information acquisition apparatus according to claim 6 or 7, wherein the detection of the electrochemical change is detection of at least one of current, charge amount, potential, voltage, and impedance.   A stress sensor comprising the information acquisition device according to claim 1 and a stress level determination unit. A stress level information acquisition device for acquiring information on a stress level of a measurement target person,
Using the redox reaction of thioredoxins in the sample derived from the measurement subject, at least one of the oxidized form concentration and the reduced form concentration of the thioredoxins is measured separately.
Measurement based on the first information selected from the oxidant concentration of thioredoxins, the concentration of the reductant, and the ratio of these concentrations, and the second information on the relationship between the first information and the degree of stress. A stress level information acquisition apparatus comprising stress level determination means for determining a stress level of a subject.   The stress level information acquisition apparatus according to claim 10, further comprising: a determination result output unit that outputs a result of the stress level determined by the stress level determination unit; and a display unit that displays the output determination result.   11. A storage means for storing the output from the determination result output means, and a change in the degree of stress over time of the measurement subject can be output from the determination result output means from the determination results accumulated in the storage means. The stress degree information acquisition device described in 1.   The stress level information according to claim 10, wherein at least one of an oxidized form concentration of thioredoxins and a concentration ratio of the oxidized form and a reduced form in the sample of the measurement subject is used for the determination of the stress level. Acquisition device. In the stress level determination method for determining the stress level of the measurement subject,
Using the redox reaction of thioredoxins in the sample from the measurement subject, the degree of stress based on at least one of the oxidized form concentration and reduced form concentration of the thioredoxins, and a preset standard A method for determining a stress level, comprising the step of determining   The stress level determination according to claim 14, wherein at least one of an oxidized form concentration of thioredoxins and a concentration ratio of the oxidized form and a reduced form in the sample of the measurement subject is used for the determination of the stress level. Method. In an enzyme electrode having a conductive member and an enzyme,
An enzyme electrode, wherein the enzyme is an enzyme that catalyzes a redox reaction of thioredoxins.   The enzyme electrode according to claim 16, wherein the enzyme is a thioredoxin reductase. In a method for measuring the concentration of thioredoxins in a sample,
A method for measuring the concentration of thioredoxins, characterized by distinguishing and measuring at least one of the oxidized form concentration and the reduced form concentration of the thioredoxins using a reaction of an oxidoreductase of thioredoxins in a sample .   The measurement method according to claim 18, wherein the enzyme is thioredoxin reductase.

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